Establishment Of Fluc Labeled HCV Cell Models And Their Application To Anti-HCV Drugs Evaluation And Other Fields

Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease. At present, HCV infection affects approximately 170 million people worldwide, more than three to four million people are newly infected each year. The majority of infected individuals develop a persistent infection, which is associated with a high risk of liver cirrhosis and hepatocellular carcinoma. HCV threats people health seriously. Today Peg-IFN and ribavirin combination therapy is the standard clinical treatment, but about 50% of patients can achieve sustained virologic response (SVR) . So development of high-effective anti-HCV drugs is of great significance.Since its discovery, progress in our understanding of this virus has been suboptimal due to the lack of good model systems. However, in the past decade this has greatly accelerated with the development of various in vitro cell model systems. These systems have made a major impact on the field of HCV research, including understanding of the mechanisms associated with HCV replication and infection and HCV pathegensis. Importantly, the in vitro cell model systems have allowed preclinical evaluation of numerous novel antiviral compounds for the treatment of chronic HCV infection.At present, research and application of HCV replication system and infectious system are hot. Base on obtained HCV subgenomic plasmid, we established and improved the firefly luciferase (Fluc) labeled HCV subgenomic replicon cell model. Base on obtained HCV full-length genome plasmid, we also reconstructed full-length genome by adding neo gene and established Fluc labeled HCV full-length genome infectious cell model. We evaluated the three drugs of IFN-α, cyclosporine A (CsA) and sorafenib on anti-HCV effect, and studied antivirus immune mechanism by using these two cell models. The contents and results are as follows:1. HCV subgenomic RNA, Luc-Ubi was prepared by in vitro transcription and then transfected into Huh7.5.1 cells with DMRIE-C transfection reagent. G418-resistant clones of Huh7.5.1 cells were obtained by selection. Luciferase activity and HCV NS5A protein were detected in selected cells harboring replicon RNA by luciferase activity assay and Western blot, respectively. The experimental results showed that Fluc labeled HCV subgenome replication cell model was established.2. Neo resistance gene was inserted into HCV full-length genome plasmid Luc-JC1 to make Luc-JC construct. Luc-JC RNA was transfected into Huh7 cells with DMRIE-C transfection reagent. G418-resistant clones of Huh7 cells were obtained by selection. Fluc, HCV core, NS3, NS5A, NS3/4A protein and RNA were identified by luciferase activity assay, Western blot, Fluorescence, and quantitative RT-PCR, respectively. The experimental results showed that Fluc labeled HCV full-length genome infectious cell model was established.3. When HCV replicon was used for evaluation of anti-HCV therapeutics, we found Fluc activity declined by increment of drugs concentration after IFN-α, CsA and sorafenib treatment. When HCV replicon was used for evaluation the anti-HCV of these three drugs, Fluc activity, protein expression level and RNA copies of HCV declined in dose-dependent manner by luciferase activity assay, Western blot and FQ-PCR. A series of experiments showed that IFN-α, CsA and sorafenib had obvious inhibition to HCV replication in dose-dependent manner. Compared their inhibition effects on HCV replicaiton, IFN-αwas the strongerest, CsA in the middle, and sorafenib the weakest. The preliminary experiments of combination therapy showed IFN-αand CsA had synergy to inhibit HCV replication. But sorafenib probably antagonize the inhibition of IFN-αto HCV replication. And the mechanism needs further study. Fluc labeled HCV replication and infectious cell models were established and their reliability and practicality was verified by drug evaluation experiments. In these cell models, Fluc reporter can be used to as an indicator of intracellular HCV replication level, which makes evaluation of anti-HCV drugs easier, simpler and faster.4. HCV replicon and infectious cell models not only can be applied in many aspects of drug evaluation and screening, but also the research of HCV pathogenesis. In vitro, we tentatively confirmed that HCV could cleave MAVS, and apparently inhibit activation of IFN-βpromoter. The mechanism of HCV NS3/4A and NS4B pariticipating in the inhibition of innate antiviral immunity was explored using a quantitative protein interaction assay. The results showed that both HCV NS3/4A and NS4B inhibited MAVS-TRAF3 interaction. Besides, NS4B may also target IRF3. In vivo, Fluc reporter activity gadually dimished, and became undetectable at short term post-transplantation of Fluc reporter HCV infectious cells into SCID-beige mice. Therefore, we speculated that innate immunity might result in inhibition of HCV infection in vivo. IFN and its activated genes expression in subcutaneously transplanted tumor were detected by RT-PCR and Western Blot. The results tentatively showed that HCV infectious cells of subcutaneous transplantation activated antivirus immune response in mice. The espression level of ISG15 in Huh7 cells of subcutaneous transplantation elevated significantly. It suggested the possible reason for activation of antivirus immune response in subcutaneous transplantated HCV infectious cells was mouse innate immunity reaction specifically to xenograft cell, but not to HCV RNA in the cell.In summary, we successfully established Fluc labeled HCV subgenomic replication and full-length genome infectious cell model in this study. These models were used to evaluate anti-HCV drugs and study on mechanism of antiviral immunity, which laid the prophase of groundwork for high throughput screening of antivirus drugs, HCV pathogenenesis and establishment of HCV cell implanted mouse model.